Pharmaceutical, biotechnology, or genomics companies use DNA analysis systems for target identification and drug screening in pharmaceutical drug discovery. In many of these systems, biomolecules (e.g., DNA, RNA, cDNA, proteins) labeled with various dyes bind to chips that offer different molecular probe counterparts for binding in different locations of the chip. A scanner is then used to read the fluorescence of these resultant surface bound molecules under illumination with suitable (most often laser) light. The scanner acts like a large field fluorescence microscope in which the fluorescent pattern caused by binding of labeled molecules is scanned on the chip. In particular, a laser induced fluorescence scanner provides for analyzing large numbers of different target molecules of interest, e.g., genes/mutations/alleles, in a biological sample.
The scanning equipment typically used for the evaluation of arrays includes a scanning fluorometer. A number of different types of such devices are commercially available from different sources, such as Axon Instruments in Union City, Calif. and Perkin Elmer of Wellesly, Mass. Analysis of the data, (i.e., collection, reconstruction of image, comparison and interpretation of data) is performed with associated computer systems and commercially available software, such as GenePix by Axon Instruments, QuantArray by Perkin Elmer or Feature Extraction by Agilent of Palo Alto, Calif.
In such scanning devices, a laser light source generates a—most often collimated—beam. The collimated beam sequentially illuminates small surface regions of known location on an array substrate. The resulting fluorescence signals from the surface regions are collected either confocally (employing the same lens used to focus the laser light onto the array) and/or off-axis (using a separate lens positioned to one side of the lens used to focus the laser onto the array). The collected signals are then transmitted through appropriate spectral filters to an optical detector. A recording device, such as a computer memory, records the detected signals and builds up a raster scan file of intensities as a function of position, or time as it relates to the position. Such intensities, as a function of position, are typically referred to in the art as “pixels” or “pixel values.”
In performing scans, a typical approach is to zigzag across a microarray slide or substrate obtaining data in a raster fashion. In doing so, it has been appreciated that very slight variation in the tilt or angle of a slide to be scanned, or variation in the planarity of the slide itself, must be accounted for in order to achieve acceptable focus and accurately obtain data on successive features.
For this purpose, known systems actuate a scanning lens assembly or the cradle/caddy carrying a slide by servomechanism(s) to bring features into focus by varying the distance between the items (in an effort to maintain a constant distance between the features being scanned and the optics). Known feedback logic controllers are used to accomplish this goal.
Two common types of electronic feedback controllers are Proportional-Integral (PI) and Proportional-Integral-Derivative (PID) controllers. The implementation of each may vary widely. Tuning and custom design of the same are well within the abilities of those with ordinary skill in the art.
The tuning required to make a selected control system suitable for a given application involves scaling the contribution to the control output of each component of the controller selected. The proportional component(s) of either type of controller operates to direct corrective action to a control element based on the present state of a given process relative to a desired setpoint. Integral component(s) operate by directing control action based on the sum of previous errors in the process. The error sum tends toward zero (and thus a desired state for a given process) as negative error conditions subtract from a positive error total or vice versa due to corrective action taken. Derivative components in a PID controller direct corrective action in response to a change in slope or sign of a measured error condition. As the derivative of a measured value is taken, this term is,keyed to rate of change of a process. Implementations of derivative control features include use in making larger or stepwise corrections as well as damping out system oscillations.
In electronic controllers as described, the measure of a given corrective effect in relation to the corrective input is understood in terms of control element gain. The controller's bias represents the control effort required to maintain the process at its setpoint absent external loading of the system.
With this understanding of the relevant controller types in mind, certain considerations in array scanning should be appreciated as background to the present invention. Namely, in typical array scanner systems, a lens is scanned back-and-forth across a slide or substrate, while a control algorithm attempts to hold focus by maintaining the distance between a lens and slide despite asymmetries present in the system. Without the teaching of the present invention, however, if the slide being scanned is steeply tilted or bowed with respect to the lens (i.e., the left side of the slide is nearer the lens than the right side, or vice versa), the inherent delays of an applicable PI or PID control algorithm cause the actual slide position to lag behind its setpoint/in-focus position. The integral term of the PI or PID control equation attempts to make up for this lag by, in effect, anticipating that the recently observed slope will continue and acting accordingly.
Generally, in a PID controller,Vout(t)=kpe(t)+k1I(t)+kdD(t)  [1]where Vout(t) is the servo control voltage output at time step t, e(t) is the position error measured at time t, I(t) is the running sum of e(t), from t=0 until t, D(t) is the derivative of e(t) and kp, kl, and kd are tuning parameters. As one might suspect, in a PI system, there is no derivative term. In either type of system, additional terms may be included to further refine matters or provide additional functionality. Other related control equations are well known in the art as well.
During scanning of a sloped surface (i.e., a surface with a distance from the focused lens that increases or decreases substantially monotonically as the scan progresses), the l(t) term in equation [1] (the “integral” term—discussed below in terms of “I” alone in connection with the present invention) will grow until it reaches a value which corrects for the amount the error changes between the time it is measured and the time the control voltage takes effect.
When the scanner reverses direction, the sign of the slope that the integral term compensates for reverses. Since the integral term continues to add to its running sum, it will eventually adapt to the new tilt direction, and the controller will again control without error. However, in known systems, for a brief period at the start of the reversed scan line (the time until the integral term has time to adapt), the system will not be able to correct for the control loop lag.
This situation usually causes noticeable focus errors for the first few millimeters of each scan line. Typically, focus is undershot, at least partly because the integral term has not had sufficient time to “grow” adapting to the new conditions in the opposite scan direction. After undercompensating for focus, the typical result is overshooting focus as the control loop compensates for the observed error in the negative. The focus error causes a dip in the signal intensity in the scanned image at the beginning of the scan line. This sudden change in the signal intensity adversely affects the uniformity specifications of the scanner.
In instances where fully adaptive focus control is feasible (meaning that where significant borders or edge portions are provided around a scan area of a slide that are situated across from the lens assembly during the full motion of the system and the system is allowed to adapt to changing slope as it turns around), the system would be in focus upon returning to the region of interest. However, it is common practice to maximize array sizing/placement on a slide or substrate, leaving no room for purely adaptive control to provide accurate focus.
Accordingly, systems have been developed, such as described in U.S. patent application Ser. No. 10/087,220, entitled “Bi-Directional Scanner Control System,” filed Feb. 28, 2001 (hereinafter the “'200 Application”) that control to or maintain focus at a desired setpoint once leaving an active scan region of a slide corresponding to where array features are to be provided. Despite the marked improvement offered by that system over previous approaches, upon returning to the scan area after completion of scanning a line and turning around, another sort of focus error is introduced. Namely, mechanical/electromechanical delays inherent to moving components against inertial loads make the system unable to—respond instantly to conditions (such as slide tilt and/or curvature) requiring focus adjustment, even in response to a signal that is otherwise adequate to set focus. Accordingly, while the system in the '220 Application takes steps to avoid issues with the control loop integral term, it still sometimes under compensates for focus and then overcompensates in a like manner in instances where the scanner's physical parameters prohibit accurately tracking the slide/array surface upon reentering an active scan region.
The present invention offers a further improved focus control approach. Control algorithm integral terms are accounted for in a manner resembling the approach in the '220 Application where they are artificially set based on the state of a preceding scan line. However, the additional transient focus errors inherent in the referenced invention are accounted for. As such, the present invention offers expanded utility in dealing with more extreme situations. The approach is suitable for more demanding applications where slide tilt and/or curvature varies greatly. Yet, it is applicable in less extreme scanning applications as well. As such, the present invention meets the continuing need for improved data acquisition.